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At least 19 recordsLinked to original sources
PubMed2023

Structural basis for pre-tRNA recognition and processing by the human tRNA splicing endonuclease complex.

Throughout bacteria, archaea and eukarya, certain tRNA transcripts contain introns. Pre-tRNAs with introns require splicing to form the mature anticodon stem loop. In eukaryotes, tRNA splicing is initiated by the heterotetrameric tRNA splicing endonuclease (TSEN) complex. All TSEN subunits are essential, and mutations within the complex are associated with a family of neurodevelopmental disorders known as pontocerebellar hypoplasia (PCH). Here, we report cryo-electron microscopy structures of the human TSEN-pre-tRNA complex. These structures reveal the overall architecture of the complex and the extensive tRNA binding interfaces. The structures share homology with archaeal TSENs but contain additional features important for pre-tRNA recognition. The TSEN54 subunit functions as a pivotal scaffold for the pre-tRNA and the two endonuclease subunits. Finally, the TSEN structures enable visualization of the molecular environments of PCH-causing missense mutations, providing insight into the mechanism of pre-tRNA splicing and PCH.

Humans↗
PubMed2024

Self-quenched tRNA reporters for imaging tRNA-derived RNA biogenesis.

tRNA-derived small RNAs (tDRs) are an emerging class of small non-coding RNAs that play crucial roles in various cellular processes. However, there is a paucity of data on their sub-cellular localization due to a lack of tools and reagents to image tDRs. Imaging tDRs remains challenging due to the similar sequences between tDR and its parent tRNA. Here, we describe an innovative tool for studying the formation and localization of tDRs in various biological processes using a self-quenched tDR biogenesis reporter. This method utilizes a full-length tRNA molecule conjugated with both fluorescence and quencher groups at 5'- and 3'- ends. In its intact state, the fluorescence is quenched. Upon cleavage by specific ribonucleases and strand separation, the fluorescence becomes detectable, allowing real-time imaging of tDR biogenesis. This protocol details the design, synthesis, and application of this reporter, including transfection procedures and imaging techniques. The method offers a powerful approach for investigating tDR dynamics in living cells, providing insights into their roles in cellular processes and stress responses.

RNA, Transfer↗
PubMed2023

Quantitative analysis of tRNA abundance and modifications by nanopore RNA sequencing.

Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress.

RNA↗
PubMed2026

tRNA methylation: functional insights and epitranscriptomic regulation.

tRNAs, one of the most conserved and abundant RNAs, are central components of protein synthesis, transferring genetic information from DNA to proteins through a precise base-pairing mechanism. Post-transcriptional modifications of tRNAs by tRNA modifying enzymes are essential for maintaining their normal physiological functions, including methylation, isomerization and glycosylation. tRNA methylation, particularly 1-methyladenosine (m1A), 5-methylcytidine (m5C), and 7-methylguanosine (m7G), are among the most abundant and diverse types of post-transcriptional modifications of tRNA, which promote the stability of tRNA secondary and tertiary structures and allow for proper translation. In addition, tRNA methylation affects the production and function of tsRNA (tRNA-derived small RNA), small fragments of RNA that further regulate gene expression and protein synthesis. In our review, we discuss the relevant biological functions of tRNA methylation, including tRNA stability, protein translation, and tsRNA biogenesis.

RNA, Transfer↗
PubMed2026

Pathogenic human mitochondrial tRNA variants impair RNA processing by compromising 5' leader removal.

Human mitochondrial genome (mtDNA) encodes multiple proteins in the oxidative phosphorylation complexes as well as the ribosomal and transfer RNAs (tRNAs) needed for in situ translation. These genes are transcribed from only three promoters, producing polycistronic transcripts that are co-transcriptionally cleaved by mitochondrial RNase enzymes to release majority of individual gene products. tRNAs separate many of these genes and are thought to serve as "punctuation" marks that enable RNase recognition, binding, and hydrolysis of the 5' "leader" and 3' "trailer" sequences flanking the tRNA. Mutations in the tRNA genes dominate the mtDNA-linked mitochondrial pathologies; yet a systematic study of the impact of tRNA sequence variation on the RNase-catalyzed processing is lacking. Here, we employed human mitochondrial tRNATyr as a model system to dissect the effect of tRNA variants on the in vitro 5' leader and 3' trailer hydrolysis. We found that nucleotide variations located near the catalytic interfaces - particularly within or near the tRNA acceptor stem - showed the strongest defects in 5' processing and prevented release of the downstream tRNA in a tRNA cluster where multiple tRNAs are transcribed in tandem. This work provides mechanistic insight into how mutations disrupt coordinated mitochondrial tRNA processing and establish a framework for predicting variant effects based on their structural position relative to the processing enzymes.

Journal Article↗
PubMed2024

TGIRT-seq to profile tRNA-derived RNAs and associated RNA modifications.

RNA modifications are key regulators for RNA processes. tRNA-derived RNAs are small RNAs with size between 15 and 50 bases long that are processed from mature or precursor tRNAs. Despite their more recent discovery, tRNA-derived RNAs have been found to play regulatory roles in many cellular processes including gene silencing, protein synthesis, stress response, and transgenerational inheritance. Furthermore, tRNA-derived RNAs are highly abundant in bodily fluids, posing as potential biomarkers. A unique feature of tRNA-derived RNAs is that they are rich in RNA modifications. Many of the RNA modifications on tRNA-derived RNAs disrupt Watson-Crick base pairing and will thus stall reverse transcriptase, such as N1-methyladenosine (m1A), N1-methylguanosine (m1G) and N2, N2-dimethylguanosine (m22G). These RNA modifications add another layer of regulation onto tRNA-derived RNAs' functions and are of interests for future research. However, these RNA modifications could also lead to lower detection of modification-containing RNAs in genome-wide small RNA sequencing analysis due to reverse transcriptase stall. To circumvent this bias, TGIRT (Thermostable Group II Intron Reverse Transcriptase) has been used to readthrough RNA modifications inserting mismatches. These mismatch signatures can then be used to precisely map the modification sites at base resolution. Here we describe the step-by-step experimental protocol to start with purified RNAs from cells or tissues and use TGIRT to make small RNA sequencing library for Illumina sequencing to profile the abundance of tRNA-derived RNAs and the associated RNA modifications.

RNA, Transfer↗
PubMed2025

Aminoacyl-tRNA Specificity of a Ligase Catalyzing Non-ribosomal Peptide Extension.

Peptide aminoacyl-transfer ribonucleic acid ligases (PEARLs) are amide-bond-forming enzymes that extend the main chain of peptides by using aminoacyl-tRNA (aa-tRNA) as a substrate. In this study, we investigated the substrate specificity of the PEARL BhaBCAla from Bacillus halodurans, which utilizes Ala-tRNAAla. By leveraging flexizyme, a ribozyme capable of charging diverse acids onto a desired tRNA, we generated an array of aa-tRNAs in which we varied both the amino acid and the tRNA to dissect the substrate scope of BhaBCAla. We demonstrate that BhaBCAla catalyzes peptide extension with noncognate proteinogenic and noncanonical amino acids, hydroxy acids, and mercaptocarboxylic acids when attached to tRNAAla. For most of these, the efficiency was considerably reduced compared to Ala, indicating that the enzyme recognizes the amino acid. By variation of the different parts of the tRNA, enzyme specificity was shown to also depend on the acceptor stem and the anticodon arm of the tRNA. These findings establish the molecular determinants of PEARL specificity and provide a foundation for engineering these enzymes for broader applications in peptide synthesis.

RNA, Transfer, Amino Acyl↗
PubMed2025

A survey of dietary effects on tRNA abundance and modifications.

Transfer RNAs (tRNAs) play a central role in protein translation and are increasingly recognized as dynamic regulators of gene expression. Both physiological and environmental signals can modulate tRNA abundance and chemical modifications, yet the impact of dietary cues on the tRNA landscape remains poorly understood. Here, we investigated the effects of two distinct dietary interventions-low-protein and high-fat diets-on tRNA abundance and modification profiles across multiple mouse tissues. We conducted a comprehensive analysis of tRNA abundance and modification changes in response to these nutritional challenges using RNA mass spectrometry and Ordered Two-Template Relay sequencing (OTTR-seq), a modified-base-sensitive tRNA sequencing method. Our results reveal both shared and tissue-specific alterations in abundance and modifications of specific nuclear and mitochondrial genome-encoded tRNAs in response to dietary conditions at isotype, isoacceptor, and isodecoder levels. As many of the tissue-specific or diet-responsive tRNA modifications have been previously reported to affect decoding efficiency or translational fidelity, these results have implications for understanding translational adaptation in response to dietary conditions.

Journal Article↗
PubMed2026

Dietary effects on cytosolic and mitochondrial tRNA abundance and modification patterns across mouse tissues.

Transfer RNAs (tRNAs) are central to protein synthesis and are increasingly recognized as dynamic regulators of gene expression whose abundance and chemical modifications are subject to precise biological control. Here, we systematically investigate how two distinct dietary interventions, low-protein and high-fat diets, reshape the tRNA landscape across multiple mouse tissues, using RNA mass spectrometry and ordered two-template relay sequencing (OTTR-seq) to comprehensively profile cytosolic and mitochondrial tRNAs at single-nucleotide resolution. We reveal pronounced tissue-specific biases in tRNA isodecoder expression, including the unexpected presence of full-length cytosolic tRNAs in mature sperm with a distinct isotype composition. In somatic tissues such as liver and heart, dietary conditions alter both tRNA abundance and key modifications known to regulate decoding efficiency, whereas in reproductive tissues diet primarily affects the abundance of select tRNAs with comparatively limited changes in modification profiles. We further demonstrate that mitochondrial tRNAs are subject to diet-responsive changes in both abundance and modification status and that even subtle differences in dietary fat composition are sufficient to alter tRNA modification signatures. Together, these findings establish the tRNA epitranscriptome as a sensitive and tissue-specific sensor of nutritional state and provide a resource for understanding how dietary cues interface with translational regulation in somatic and reproductive tissues.

Male↗
PubMed2025

A hypoxia-responsive tRNA-derived small RNA confers renal protection through RNA autophagy.

Transfer RNA-derived small RNAs (tsRNAs or tDRs) perform a range of cellular functions. Here, we showed that tRNA-Asp-GTC-3'tDR, a hypoxia-induced tDR derived from the 3' end of tRNA-Asp-GTC, activated autophagic flux in kidney cells and its silencing blocked autophagic flux. Functional gain-/loss-of-function studies in murine kidney disease models demonstrated a substantial renoprotective function of tRNA-Asp-GTC-3'tDR. Mechanistically, tRNA-Asp-GTC-3'tDR assembled stable G-quadruplex structures and sequestered pseudouridine synthase 7 (PUS7), preventing catalytic pseudouridylation of histone mRNAs. The resulting pseudouridylation deficiency directed histone mRNAs to the autophagosome-lysosome pathway, triggering RNA autophagy. This tDR-induced RNA autophagy pathway was activated during murine and human kidney diseases, suggesting clinical relevance. Thus, tRNA-Asp-GTC-3'tDR plays a role in regulating RNA autophagy, which helps to maintain homeostasis in kidney cells and protects against kidney injury.

Animals↗
PubMed2026

Translational reprogramming of TGF-β signaling via TRMT61A-mediated tRNA m1A drives prostatic fibrosis and hyperplasia.

Dysregulation of the epitranscriptomic landscape is closely linked to pathological proliferation, but its specific role in benign prostatic hyperplasia (BPH) remains unclear. Here, we identify the tRNA methyltransferase TRMT61A as a critical driver of BPH progression. We found that TRMT61A and global N1-methyladenosine (m1A) levels are aberrantly upregulated in human BPH tissues. Functionally, TRMT61A knockdown potently suppresses prostate cell proliferation and reduces stromal fibrosis, inducing G1 cell cycle arrest and reversing pathological remodeling both in vitro and in vivo. By integrating ribosome profiling (Ribo-seq) and tRNA-seq, we observed that TRMT61A drives translational reprogramming. TRMT61A preserves the stability of specific tRNA isoacceptors (e.g., tRNA-Leu-CAA), which is required for the efficient decoding of mRNAs containing m1A-dependent codons. Consequently, TRMT61A selectively promotes the translational elongation of the key receptor TGFβR1. This amplifies downstream TGF-β/SMAD signaling and drives epithelial-mesenchymal transition (EMT) without affecting mRNA transcription. In summary, our study reveals how TRMT61A drives BPH progression through TGFβR1 translation, highlighting the therapeutic potential of targeting epitranscriptomic pathways to reverse prostatic hyperplasia and fibrosis.

Male↗
PubMed2026

The halophilic archaeon Halogranum roseipondis sp. nov. is susceptible to a virus carrying an exceptionally high number of viral tRNA genes.

UNLABELLED: Archaea constitute a diverse group of organisms, many of which inhabit extreme environments, such as haloarchaea that dominate hypersaline ecosystems, like solar salterns. Sampling of solar salterns and other hypersaline environments has resulted in numerous haloarchaeal isolates, including 3 classified and 27 uncharacterized Halogranum species. However, no complete genome has so far been reported for any member of this genus. Here, we present the first comprehensive study of Halogranum sp. SS5-1 isolated from a solar saltern in Samut Sakhon, Thailand. Hgn. SS5-1 is a pleomorphic, aerobic heterotroph that thrives in high salinity and moderate temperature and is capable of hydrolyzing starch. Its genome consists of a 3.6 Mbp chromosome and seven additional plasmids. Based on our phylogenetic analyses, which establish Hgn. SS5-1 as a distinct species, we propose that it will be classified as the novel species Halogranum roseipondis sp. nov. SS5-1T. Additionally, we report that Hgn. roseipondis sp. nov. SS5-1T is infected by Hagravirus capitaneum (HGTV-1), the only virus known to infect a Halogranum host. HGTV-1 exhibits a unique head-tailed morphology and encodes the largest archaeal virus double-stranded DNA genome known to date, including 34 tRNA-encoding genes. Codon usage analysis of the viral genome suggests partial alignment with host preferences, yet the abundance of viral tRNA genes hints at broader roles, potentially including roles in translation and host regulation. This study establishes Hgn. roseipondis and HGTV-1 as a novel virus-host system, opening avenues to explore infection dynamics and the roles of virus-encoded tRNA in archaea. IMPORTANCE: Archaea that thrive in high-salinity environments are key players in geochemical cycles and important contributors to ecosystem productivity. Despite their ecological significance and importance for the development of novel methodologies in synthetic biology, haloarchaea remain poorly studied. Further exploration of haloarchaea is required to obtain valuable information on the evolution of cellular complexity and the molecular mechanisms that allow cells to thrive in harsh environmental conditions. Here, we present the characterization of a novel archaeon, Halogranum roseipondis sp. SS5-1T, alongside the infection cycle of its associated virus, Hagravirus capitaneum. This tailed myovirus carries an extraordinary set of 34 viral tRNA genes, a feature that opens intriguing questions about virus-host interactions and translational control. Our findings lay the groundwork for future investigations into the expression and function of viral tRNAs in an archaeal model system, thereby opening a new frontier for studying archaeal translation and virus-driven modulation of host cellular processes.

Halobacteriaceae↗
PubMed2026

Pyridoxal-phosphate-dependent biosynthesis of aminovaleramide by AvaS in tRNA.

In eubacteria, decoding of isoleucine codon AUA requires a specialized tRNA (tRNAIle2) modified with lysidine (k2C) at the anticodon wobble position (C34), which switches decoding specificity from methionine (AUG) to isoleucine (AUA). Recently, aminovaleramide cytidine (ava2C) was discovered at the same tRNA position in several bacteria and plants and shown to support AUA decoding and Ile-specific aminoacylation. However, the enzyme catalyzing ava2C was unknown. Here, we report that tRNAIle-aminovaleramididine synthetase (AvaS) catalyzes ava2C biosynthesis in Pseudomonas aeruginosa PA14. AvaS converts k2C to ava2C through a pyridoxal-phosphate-dependent oxidative decarboxylation mechanism, supported by site-directed mutagenesis and in vitro enzymatic assays. Dual-reporter assays demonstrated that ava2C-modified tRNA exhibits lower AUA decoding efficiency than k2C-modified tRNA. Additionally, genome-wide screening revealed an unexpected link between ava2C levels and metabolic and stress response pathways influencing i6A/ms2i6A dynamics. Together, these findings define the molecular basis of ava2C biosynthesis and its broader cellular metabolic networks.

Journal Article↗
PubMed2026

tRNA m1A modification orchestrates STING translation in macrophages to enhance antitumor immunity and CAR-macrophage immunotherapy.

Tumor-associated macrophages (TAMs) play crucial roles in tumor progression. However, the mechanisms underlying the posttranscriptional regulation of TAMs remain largely unknown. Here, we demonstrated that Trmt61a, the "writer" enzyme of tRNA N1-methyladenosine (m1A) modification, is highly expressed in proinflammatory macrophages in tumor microenvironment. We generated conditional knockout (KO) mice for Trmt61a and observed that Trmt61a deletion in macrophages significantly promoted tumor growth. Mechanistically, we identified that m1A maintains the translation of STING, enhances STING-TBK1-IFN-β signaling in macrophages and therefore suppresses tumor cell growth. We further generated TRMT61A-overexpressing human iPSC-derived CAR-macrophage and demonstrated that human TRMT61A effectively promoted antitumor CAR-macrophage therapy in vivo. Collectively, our findings reveal a novel regulatory mechanism of tRNA m1A modification in macrophages, highlighting the antitumor therapeutic potential of targeting tRNA m1A modification in macrophages.

Animals↗
PubMed2026

Involvement of tRNA thiolation in uORF-mediated translational regulation during Xylogenesis in Arabidopsis thaliana.

Post-transcriptional modification of tRNAs is an important mechanism for regulating translation efficiency and cellular homeostasis, yet its contribution to upstream open reading frame (uORF)-mediated translational control remains largely unexplored. In this study, we investigated the role of tRNA thiolation in thermospermine-dependent regulation of xylem development in Arabidopsis thaliana. Using a suppressor screen of the thermospermine-deficient mutant acaulis5 (acl5), which exhibits dwarfism and excessive xylem differentiation, we identified suppressor-of-acl502 (sac502) as a recessive loss-of-function allele of CTU2, a gene encoding a key enzyme in the biosynthesis of the wobble uridine modification 5-methoxycarbonylmethyl-2-thiouridine. Mutations in other components of the same modification pathway, including ROL5 and TRM9, similarly suppressed the acl5 phenotype. Translational analyses using 5' leader-GUS reporter constructs revealed that the ctu2 mutation did not enhance translation of the mRNA containing a thermospermine-responsive uORF of SAC51, but instead significantly reduced translation of that of SACL3, a member of the SAC51 family, and that of LONESOME HIGHWAY (LHW), which contains another conserved uORF in the 5' leader region. Polysome profiling further demonstrated decreased association of SACL3 and LHW mRNAs with actively translating ribosomes in ctu2. Genetic interaction analyses supported the conclusion that the suppression of excessive xylem formation in acl5 by ctu2 is attributable to reduced LHW activity. In addition, ctu2 mutants displayed increased sensitivity to exogenous thermospermine, resembling the response of lhw mutants. Together, our results reveal that tRNA thiolation contributes to uORF-mediated translational regulation of key developmental regulators and identify tRNA modification as an important regulatory layer controlling vascular development.

Arabidopsis↗
PubMed2026

One-time duplication and ongoing loss of mitochondrial tRNA genes in Cryptocercus cockroaches.

Mitochondrial genome is a popular marker in phylogenetics and species diversity estimations. Mitogenome is relatively compact and conserved, while gene rearrangements were found in some species across various organisms. Models to explain the origin and evolution of gene rearrangement have been proposed but seldom demonstrated; empirical evidence from closely related species is particularly scarce. Here, through an intensive case study of the cockroach genus Cryptocercus Scudder, 1862, we elucidate the evolution of mitochondrial gene order. This study utilized 51 new samples and re-assembled raw reads of 26 published samples. A diversity of rearrangement patterns is recovered, especially in the tRNA gene cluster between ND3 and ND5, which is effectively explained by the duplication - random loss model. Specifically, the entire tRNA gene cluster was duplicated; this duplication is potentially facilitated by chance binding between the 3' end of ND5 gene and the ND3-trnA region during DNA replication. Furthermore, we reveal that one of the gene copies degenerated stochastically across lineages, directly contributing to the observed diversity in gene arrangement. Gene rearrangement patterns are apomorphies for certain clades, providing additional evidence for the inferred phylogeny and serving as potential indicators of species. This study underscores the importance of intensive sampling and rigorous data curation for deciphering the evolutionary mechanisms.

Duplication–random loss model↗
PubMed2026

Histidine 62 in ArfB is required for stop codon-independent peptidyl-tRNA hydrolysis on the stalled ribosome.

Translating ribosomes can stall on mRNA for various reasons, including nuclease cleavage, arrest peptide sequences or ribosome collisions. In Escherichia coli, several ribosome rescue factors act to release ribosomes stalled at the 3' end of mRNA. Among these factors, ArfB can rescue stalled ribosomes without the help of other factors. ArfB consists of an N-terminal domain containing the catalytic GGQ motif, which mediates peptidyl-tRNA hydrolysis, and a C-terminal extension that functions as a sensor for recognizing stalled ribosomes. However, how these two regions coordinate to resolve ribosome stalling remains unclear. Here, using a reconstituted translation system, we found the functional importance of histidine residues in the N-terminal domain of ArfB. In particular, histidine at position 62 in E. coli ArfB was required for stop codon-independent fMet-tRNA hydrolysis, whereas its substitution did not affect affinity for the ribosome. Furthermore, directed hydroxyl radical probing revealed that H62A mutation did not significantly alter the overall positioning of either the N-terminal domain or the C-terminal extension of ArfB on the ribosome. These findings suggest that H62 contributes to ArfB function during a step following initial ribosome binding, thereby facilitating peptidyl-tRNA hydrolysis.

Ribosomes↗
PubMed2025

A hormone-dependent tRNA half promotes cell cycle progression via destabilization of p21 mRNA.

tRNA halves are among the most abundant short non-coding RNAs in the cellular transcriptome. Here we report that in androgen receptor-positive LNCaP prostate cancer cells, the hormone-dependent 5'-tRNALysCUU half promoted cell proliferation by facilitating cell cycle progression. Global mRNA profiling upon the 5'-tRNALysCUU half depletion revealed that the mRNA of p21, a negative regulator of the cell cycle, is post-transcriptionally destabilized via a 5'-tRNALysCUU half-driven mechanism. YBX1, identified as a protein interacting with 5'-tRNALysCUU half in the cytosol, was shown to stabilize p21 mRNA. Specific sequences resembling the 5'-tRNALysCUU half, located in the 3'-UTR of p21 mRNA and termed LL588, were identified as the binding site for YBX1 and are required for p21 mRNA stability. In vitro binding assays demonstrated that the 5'-tRNALysCUU half is capable of displacing YBX1 from LL588. Collectively, our findings suggest that the 5'-tRNALysCUU half directly binds to and displaces YBX1 from p21 mRNA, leading to the destabilization of p21 mRNA and the promotion of cell cycle progression in hormone-dependent cancers. Our study illuminates the role of tRNA halves in regulating mRNA stability and suggests that this may be part of broader regulatory networks affecting mRNA levels, orchestrated by various tRNA halves and their interacting proteins.

Humans↗